Phenotypic Analysis of Arabidopsis Mutants: Bacterial Pathogens

Abstract

The most commonly used bacterial pathogen of Arabidopsis is Pseudomonas syringae, pathovar tomato strain DC3000 or pathovar maculicola strain ES4326. Plants used for infection with P. syringae should be grown on a short-day light cycle, so that they develop large leaves. For consistent results, it is important that the plants are watered well and do not experience any abiotic stresses. The plants should be grown and tested in a temperature- and humidity-controlled growth chamber, because the extent of bacterial growth is highly dependent on both factors. Gene-for-gene resistance to P. syringae is usually accompanied by the hypersensitive response (HR), a form of localized cell death that occurs in response to pathogen challenge. P. syringae strains carrying one of the avirulence genes avrRpt2, avrRpm1, avrB, avrPphB, and avrRps4 trigger the HR in wild-type Columbia. Other ecotypes may lack one or more of the R genes needed for recognition of these avirulence genes. Bacterial growth can be monitored by grinding up infected tissue, plating serial dilutions on King's B medium, and counting colonies. This protocol describes methods for preparing bacterial cultures, inoculating plants, testing the HR, and assessing bacterial growth.